Nicole S. Sampson

Mediation of sperm-egg fusion: evidence that mouse egg α6β1 integrin is the receptor for sperm fertilinβ

Introduction A key step leading to fertilization is the binding of sperm to the egg plasma membrane. When a mammalian sperm reaches the egg plasma membrane, fertilinβ, an extracellular sperm membrane protein, is believed to bind to an egg plasma membrane receptor triggering fusion. We set out to identify the fertilinβ binding partner on the egg plasma membrane. Results We synthesized an 125 I-labeled peptide with the consensus Asp-Glu-Cys-Asp (DECD) sequence of fertilinβs disintegrin domain. This peptide contains a benzophenone photoaffinity probe and inhibits sperm-egg fusion. Upon photoactivation in the presence of whole mouse eggs, a sing e polypeptide was covalently labeled. This polypeptide has been identified by immunoprecipitation as an α 6 integrin complexed with β 1 integrin. Conclusions Our experiments establish that small peptides containing the consensus DECD sequence of sperm fertilinβ bind specifically to an α 6 β 1 integrin receptor on the egg membrane. We conclude that fertilinβ binds directly to the α 6 β 1 integrin on the egg surface and this partnership mediates sperm-egg fusion.

Inhibition of matrix metalloproteinase-14 (MMP-14)-mediated cancer cell migration

Matrix metalloproteinases (MMPs) have been shown to be key players in both extracellular matrix remodeling and cell migration during cancer metastasis. MMP-14, a membrane-anchored MMP, in particular, is closely associated with these processes. The hemopexin (PEX) domain of MMP-14 has been proposed as the modulating region involved in the molecular cross-talk that initiates cell migration through homodimerization of MMP-14 as well as heterodimerization with the cell surface adhesion molecule CD44. In this study, minimal regions required for function within the PEX domain were investigated through a series of substitution mutations. Blades I and IV were found to be involved in cell migration. We found that blade IV is necessary for MMP-14 homodimerization and that blade I is required for CD44 MMP-14 heterodimerization. Cross-talk between MMP-14 and CD44 results in phosphorylation of EGF receptor and downstream activation of the MAPK and PI3K signaling pathways involved in cell migration. Based on these mutagenesis analyses, peptides mimicking the essential outermost strand motifs within the PEX domain of MMP-14 were designed. These synthetic peptides inhibit MMP-14-enhanced cell migration in a dose-dependent manner but have no effect on the function of other MMPs. Furthermore, these peptides interfere with cancer metastasis without affecting primary tumor growth. Thus, targeting the MMP-14 hemopexin domain represents a novel approach to inhibit MMP-14-mediated cancer dissemination.

A Thiolase of Mycobacterium tuberculosis Is Required for Virulence and Production of Androstenedione and Androstadienedione from Cholesterol

ABSTRACT Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen that shifts to a lipid-based metabolism in the host. Moreover, metabolism of the host lipid cholesterol plays an important role in M. tuberculosis infection. We used transcriptional profiling to identify genes transcriptionally regulated by cholesterol and KstR (Rv3574), a TetR-like repressor. The fadA5 (Rv3546) gene, annotated as a lipid-metabolizing thiolase, the expression of which is upregulated by cholesterol and repressed by KstR, was deleted in M. tuberculosis H37Rv. We demonstrated that fadA5 is required for utilization of cholesterol as a sole carbon source in vitro and for full virulence of M. tuberculosis in the chronic stage of mouse lung infection. Cholesterol is not toxic to the fadA5 mutant strain, and, therefore, toxicity does not account for its attenuation. We show that the wild-type strain, H37Rv, metabolizes cholesterol to androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) and exports these metabolites into the medium, whereas the fadA5 mutant strain is defective for this activity. We demonstrate that FadA5 catalyzes the thiolysis of acetoacetyl-coenzyme A (CoA). This catalytic activity is consistent with a β-ketoacyl-CoA thiolase function in cholesterol β-oxidation that is required for the production of androsterones. We conclude that the attenuated phenotype of the fadA5 mutant is a consequence of disrupted cholesterol metabolism that is essential only in the persistent stage of M. tuberculosis infection and may be caused by the inability to produce AD/ADD from cholesterol.

Sub-atomic resolution crystal structure of cholesterol oxidase: what atomic resolution crystallography reveals about enzyme mechanism and the role of the FAD cofactor in redox activity.

The crystal structure of cholesterol oxidase, a 56kDa flavoenzyme was anisotropically refined to 0.95A resolution. The final crystallographic R-factor and R(free) value is 11.0% and 13.2%, respectively. The quality of the electron density maps has enabled modeling of alternate conformations for 83 residues in the enzyme, many of which are located in the active site. The additional observed structural features were not apparent in the previous high-resolution structure (1.5A resolution) and have enabled the identification of a narrow tunnel leading directly to the isoalloxazine portion of the FAD prosthetic group. The hydrophobic nature of this narrow tunnel suggests it is the pathway for molecular oxygen to access the isoalloxazine group for the oxidative half reaction. Resolving the alternate conformations in the active site residues provides a model for the dynamics of substrate binding and a potential oxidation triggered gating mechanism involving access to the hydrophobic tunnel. This structure reveals that the NE2 atom of the active site histidine residue, H447, critical to the redox activity of this flavin oxidase, acts as a hydrogen bond donor rather than as hydrogen acceptor. The atomic resolution structure of cholesterol oxidase has revealed the presence of hydrogen atoms, dynamic aspects of the protein and how side-chain conformations are correlated with novel structural features such as the oxygen tunnel. This new structural information has provided us with the opportunity to re-analyze the roles played by specific residues in the mechanism of the enzyme.

Role of the Hemopexin domain of Matrix Metalloproteinases in Cell Migration

The biological functions of matrix metalloproteinases (MMPs) extend beyond extracellular matrix degradation. Non‐proteolytic activities of MMPs are just beginning to be understood. Herein, we evaluated the role of proMMPs in cell migration. Employing a Transwell chamber migration assay, we demonstrated that transfection of COS‐1 cells with various proMMP cDNAs resulted in enhancement of cell migration. Latent MMP‐2 and MMP‐9 enhanced cell migration to a greater extent than latent MMP‐1, ‐3, ‐11 and ‐28. To examine if proteolytic activity is required for MMP‐enhanced cell migration, three experimental approaches, including fluorogenic substrate degradation assay, transfection of cells with catalytically inactive mutant MMP cDNAs, and addition of hydroxamic acid‐derived MMP inhibitors, were employed. We demonstrated that the proteolytic activities of MMPs are not required for MMP‐induced cell migration. To explore the mechanism underlying MMP‐enhanced cell migration, structure‐function relationship of MMP‐9 on cell migration was evaluated. By using a domain swapping approach, we demonstrated that the hemopexin domain of proMMP‐9 plays an important role in cell migration when examined by a transwell chamber assay and by a phagokinetic migration assay. TIMP‐1, which interacts with the hemopexin domain of proMMP‐9, inhibited cell migration, whereas TIMP‐2 had no effect. Employing small molecular inhibitors, MAPK and PI3K pathways were found to be involved in MMP‐9‐mediated cell migration. In conclusion, we demonstrated that MMPs utilize a non‐proteolytic mechanism to enhance epithelial cell migration. We propose that hemopexin homodimer formation is required for the full cell migratory function of proMMP‐9. J. Cell. Physiol. 217: 643–651, 2008.

Synthesis of Copolymers by Alternating ROMP (AROMP)

The alternating polymerization of cyclobutene 1-carboxylic esters and cyclohexene derivatives with the precatalyst [(H(2)IMes)(3-Br-pyr)(2)Cl(2)Ru=CHPh] is described. This reaction is synthetically accessible and provides (AB)(n) heteropolymers with an alternating backbone and alternating functionality. The regiocontrol of heteropolymer formation derives from the inability of the cyclobutene ester and cyclohexene monomers to undergo homopolymerization in combination with the favorable kinetics of cross polymerization.

Cholesterol Metabolism Increases the Metabolic Pool of Propionate in Mycobacterium tuberculosis

Mycobacterium tuberculosis can metabolize cholesterol to both acetate and propionate. The mass of isolated phthiocerol dimycoserate, a methyl-branched fatty acylated polyketide, was used as a reporter for intracellular propionate metabolic flux. When M. tuberculosis is grown using cholesterol as the only source of carbon, a 42 amu increase in average phthiocerol dimycoserate molecular weight is observed, consistent with the cellular pool of propionate and, thus, methylmalonyl CoA increasing upon cholesterol metabolism. In contrast, no shift in phthiocerol dimycoserate molecular weight is observed upon supplementation of medium containing glycerol and glucose with cholesterol. We conclude that cholesterol is a significant source of propionate only in the absence of sugar carbon sources.

Role of Matrix Metalloproteinase-9 Dimers in Cell Migration DESIGN OF INHIBITORY PEPTIDES

Non-proteolytic activities of matrix metalloproteinases (MMPs) have recently been shown to impact cell migration, but the precise mechanism remains to be understood. We previously demonstrated that the hemopexin (PEX) domain of MMP-9 is a prerequisite for enhanced cell migration. Using a biochemical approach, we now report that dimerization of MMP-9 through the PEX domain appears necessary for MMP-9-enhanced cell migration. Following a series of substitution mutations within the MMP-9 PEX domain, blade IV was shown to be critical for homodimerization, whereas blade I was required for heterodimerization with CD44. Blade I and IV mutants showed diminished enhancement of cell migration compared with wild type MMP-9-transfected cells. Peptides mimicking motifs in the outermost strands of the first and fourth blades of the MMP-9 PEX domain were designed. These peptides efficiently blocked MMP-9 dimer formation and inhibited motility of COS-1 cells overexpressing MMP-9, HT-1080, and MDA-MB-435 cells. Using a shRNA approach, CD44 was found to be a critical molecule in MMP-9-mediated cell migration. Furthermore, an axis involving a MMP-9-CD44-EGFR signaling pathway in cell migration was identified using antibody array and specific receptor tyrosine kinase inhibitors. In conclusion, we dissected the mechanism of pro-MMP-9-enhanced cell migration and developed structure-based inhibitory peptides targeting MMP-9-mediated cell migration.

Antibacterial Studies of Cationic Polymers with Alternating, Random and Uniform Backbones

Antibacterial polymers have potential as pharmaceuticals and as coatings for implantation devices. The design of these materials will be optimized when we have a complete understanding of the structural features that impart activity toward target organisms and those that are benign with respect to the mammalian host. In this work, four series of polymers in which cationic and hydrophobic groups were distributed along the backbone were tested against six different bacterial species (both Gram-positive and Gram-negative) and for host cytotoxicities (red blood cell lysis). The most effective of the polymers studied are regularly spaced, featuring a 6-8 carbon stretch along the backbone between side chains that present positively charged groups. They cause potassium efflux, disorder the bacterial cytoplasmic membrane, and disrupt the membrane potential. These polymers, available from alternating ring-opening metathesis polymerization (AROMP), offer proof of principle for the importance of regular spacing in antibacterial polymers and for the synthesis of additional functional materials based on regularly spaced scaffolds.

Pathway Profiling in Mycobacterium tuberculosis ELUCIDATION OF CHOLESTEROL-DERIVED CATABOLITE AND ENZYMES THAT CATALYZE ITS METABOLISM

Background: Cholesterol metabolism is critical in the chronic phase of Mycobacterium tuberculosis infection. Results: A cholesterol metabolite structure and an enzyme activity responsible for its degradation were determined. Conclusion: The igr operon encodes the enzymes that catalyze the final three steps in cholesterol side-chain degradation. Significance: Insight into the function of enzymes encoded in the igr operon is important for understanding the role of cholesterol metabolism in pathogenesis. Mycobacterium tuberculosis, the bacterium that causes tuberculosis, imports and metabolizes host cholesterol during infection. This ability is important in the chronic phase of infection. Here we investigate the role of the intracellular growth operon (igr), which has previously been identified as having a cholesterol-sensitive phenotype in vitro and which is important for intracellular growth of the mycobacteria. We have employed isotopically labeled low density lipoproteins containing either [1,7,15,22,26-14C]cholesterol or [1,7,15,22,26-13C]cholesterol and high resolution LC/MS as tools to profile the cholesterol-derived metabolome of an igr operon-disrupted mutant (Δigr) of M. tuberculosis. A partially metabolized cholesterol species accumulated in the Δigr knock-out strain that was absent in the complemented and parental wild-type strains. Structural elucidation by multidimensional 1H and 13C NMR spectroscopy revealed the accumulated metabolite to be methyl 1β-(2′-propanoate)-3aα-H-4α-(3′-propanoic acid)-7aβ-methylhexahydro-5-indanone. Heterologously expressed and purified FadE28-FadE29, an acyl-CoA dehydrogenase encoded by the igr operon, catalyzes the dehydrogenation of 2′-propanoyl-CoA ester side chains in substrates with structures analogous to the characterized metabolite. Based on the structure of the isolated metabolite, enzyme activity, and bioinformatic annotations, we assign the primary function of the igr operon to be degradation of the 2′-propanoate side chain. Therefore, the igr operon is necessary to completely metabolize the side chain of cholesterol metabolites.